Preparation of 99m Tc radiopharmaceuticals

ABSTRACT

Compounds of the formula I: 
     
         R+[.sup.99m TcNX.sub.4 ]-                                  I 
    
     wherein R+ represents a cation and X represents a halo group, are prepared by reaction with an azide compound in the presence of a hydrohalic acid. The compounds are useful in production of  99m  Tc-labelled radio- pharmaceuticals by reaction with ligands and monoclonal antibodies or antibody fractions.

This invention relates to the preparation of radiopharmaceuticals, andin particular to the preparation of technetium-99m (^(99m) Tc)--labelledradiopharmaceuticals.

Radiopharmaceuticals are diagnostic or therapeutic agents by virtue ofthe physical properties of their constituent radionuclides. Thus, theirutility is not based on any pharmacologic action. Most clinically useddrugs of this class are diagnostic agents incorporating a gamma-emittingnuclide which, because of its physical or metabolic properties,localizes in a specific organ after intravenous injection. Imagesreflecting organ structure or function are then obtained by means of ascintillation camera that detects the distribution of ionizing radiationemitted by the radioactive drug. The principal isotope currently used inclinical diagnostic nuclear medicine is reactor-produced metastabletechnetium-99m.

Many methods have been described for the reduction of pertechnetate(^(99m) TC^(VII) O₄ ⁻) in the preparation of ^(99m)Tc-radiopharmaceuticals. Reducing agents which have been used includestannous ion, electrolysis, ferrous ion, ferrous-ascorbate, formamidinesulphinic acid and sodium borohydride (Deutsch et a., 1983). Theselabelling procedures generally lead to the reduction of technetium tothe Tc(IV) or Tc(V) oxidation state. In many cases the compound preparedcontains the TcO moiety (Deutsch, 1979). Because of problems experiencedwith these reducing agents, the use of a substitution route for theproduction of ^(99m) Tc-radipharmaceuticals has been advocated (Deutsch& Barnett, 1980). The agents normally used for substitution reactionsare TcOX₅ ²⁻ and TcX₆ ⁶⁻ (X=Cl, Br) in which technetium is in the Tc^(V)and Tc^(IV) valency states respectively.

The present inventors have investigated the preparation of ^(99m)Tc-radiopharmaceuticals containing the TcN moiety, and have discoveredthat the TcN moiety is extremely stable to hydrolysis and that thenitrido group remains firmly attached to the Tc atom throughout a numberof substitution reactions.

According to the present invention, there is provided a novel group ofcompounds containing the TcN moiety, as well as methods for thepreparation thereof and methods for the preparation of ^(99m)Tc-radiopharmaceuticals utilizing these compounds.

According to a first aspect of the present invention, there are providedcompounds of the formula I:

    R.sup.+ [.sup.99m TcNX.sub.4 ].sup.-                       I

wherein R⁺ is a cation, preferably a soluble cation such as sodium oranother alkali metal, or ammonium, and X represents a halogen group,particularly a chloro or bromo group.

The compounds of this aspect of the invention are characterized by thepresence of the nitridotetrahalotechnetium-99m anion in which Tc is inthe Tc^(VI) valency state, and which has been found to have particularutility in the preparation of radiopharmaceuticals containing the TcNmoiety.

In another aspect of this invention, there is provided a process for thepreparation of compounds of the formula I as described above, whichcomprises reaction of a compound containing the ^(99m) Tc-pertechnetateanion (R[^(99m) TcO₄ ], wherein R represents a cation such as alkalimetal, or ammonium), with an azide compound, such as sodium azide, inthe presence of a hydrohalic acid, such as hydrochloric or hydrobromicacid.

In another aspect, there is provided a method of producing a ^(99m)Tc-labelled product, which comprises reacting a compound of the formulaI with a ligand. Suitable ligands includes, for example, methylenediphosphonate (MDP), thiourea (TU), thiomalate (TMA),dimercaptosuccinate (DMSA), gluconate (GLUC),N-(2,6-diisopropylphenylcarbamoylmethyl)iminodiacetate (PIPIDA),N-(2,6-dimethylphenylcarbamoylmethyl)iminodiacetate (HIDA)ethane-1-hydroxy-1,1-diphosphonate (EHDP),diethylenetriaminepentaacetate (DTPA), and cysteine (CYS). Other ligandswhich may be used in accordance with the present invention includethiouracil, diethyldithiocarbamate, mercaptopyridine,mercaptopyrimidine, thiooxine, acetylacetone, pyridoxal, oxine,tropolone and tetracycline. Monoclonal antibodies which may also belabelled in accordance with the present invention, and which have beenshown to retain their specificity following labelling. This aspect ofthe invention is described in greater detail hereinafter.

In yet another aspect, there is provided a ^(99m) Tc-labelled productwhich comprises the reaction product of a compound of formula I with aligand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1, 2, and 3 represent UV spectral studies.

FIG. 4 represents animal distribution studies.

The desirability of using a substitution route for the preparation of^(99m) Tc-radiopharmaceuticals has long been recognized. However themethod has suffered because of the difficulty in obtaining ^(99m) Tc ina suitable chemical form at the Tc concentrations used forradiopharmaceutical production. The nitrido labelling techniquedescribed here is a comparatively simple method for the preparation of awide range of radiopharmaceuticals based on the TcN moiety. While Tc isinitially present as Tc(VI) in TcNCl₄ ⁻, it has been found thatreduction usually takes place to the Tc(V) state if the ligand has theability to act as a reducing agent. Ligand substitution then takes placearound the TcN²⁺ core. In all cases studied to date, the presence of thenitrido group has been found to alter the biological behaviour of the^(99m) Tc-labelled ligand. Nitrido labelling has been found to beparticularly suitable for the labelling of "soft" ligands.

The present invention has particular application in the coupling of^(99m) Tc to monoclonal antibodies (MAb) and the use of the resultingcomplexes, for example, in specific the detection of tumors in vivo,(see, for example, Rhodes, et al, 1982). At present a number ofdifficulties exist in the diagnostic radiolocalization of tumors, one ofwhich is the choice of radionuclide. Many studies have used 131I,however this radionuclide has serious drawbacks including a poor qualityimage, significant radiation exposure due to its beta emissions, andshort biological half-life.

Technetium -99m (^(99m) Tc) as an isotope for radio-localization offersseveral advantages: it has a reasonably short half-life; it is cheap,easy to produce, and is readily available. The isotope has an optimalgamma energy (140 keV) for detection with currently available gammascintigraphic instrumentation and produces very little radiationexposure to patients undergoing scanning procedures. However little usehas been made of ^(99m) Tc for labelling antibodies, presumably becausemost labelling methods used to date lead to loss of antibody activity,due to side reactions taking place and to transchelation reactionsoccurring in vivo.

In accordance with the present invention, compounds of the formula Idescribed above have been found to produce stable ^(99m) Tc-labelled MAbby a substitution reaction in the same manner as other ligands describedherein. Either whole monoclonal antibodies, or antibody fragments (suchas Fab fragments) which react with the corresponding antigens may belabelled in accordance with this invention. Antibody specificity ismaintained in the labelling process and the labelled product is stable.In addition, tests have shown that when the labelled MAb is used intumor detection, tumors may be visualized in as soon as two hours, andfurthermore small tumors (about 0.4 cm) located near large vascularorgans can be visualized.

Preferably, in labelling monoclonal antibody in accordance with thisinvention, the antibody is at least partially reduced for example byreaction with a reducing agent such as dithiothreitol, in order toconvert disulfide linkages into sulfhydryl residues. Such a partialreduction procedure enables utilization of the presence of the TcNmoiety for sulfur atoms, and thus enables the production of more stablecomplexes in which the MAb ligand is bound through sulfur atoms to theTcN moiety. It is also noted that these sulfhydryl groups would beremoved from the sites responsible for antibody specificity, henceformation of the complexes is less likely to cause loss of specificity.

The following Examples illustrate the preparation of [^(99m) TcNX₄ ]⁻compound as well as the biological behaviour of TcN radiopharmaceuticalscontaining various ligands.

EXAMPLE 1

A. Preparation of sodium tetrachloronitridotechnetate Na[^(99m) TcNCl₄ ]and ^(99m) TcN-radipharmaceuticals.

Unless otherwise stated all solvents and chemicals were of analyticalgrade. L(+)-Cysteine for biochemistry was obtained from E. Merck,Darmstadt, Methylenediphosphonic acid (MDP) from Sigma Chemical Co., St.Louis and diethylenetriaminepentaacetic acid (DTPA) from Koch-LightLaboratories, Colnbrook. Sodium-2-gluconate was obtained from Fluka A.G. EHDP was prepared using the method described by Castronovo (1974).

HIDA and PIPIDA hydrochlorides were prepared by the reaction of theN-chloroacetanilides with iminodiacetic acid using a variation of themethod of Callery et al (1976). A mixture of N-chloroacetanilide (0.05mole), iminodiacetic acid (0.05 mole) and 10 g anhydrous sodiumcarbonate was refluxed in 30 mL of 75% aqueous ethanol for 6 hours. Oncooling the solution was acidified with concentrated hydrochloric acidand the pH adjusted to 1.5. The precipitate was filtered andrecrystallised from 50% aqueous ethanol. Use of sodium carbonateresulted in improved yields (>60%). ⁹⁹ Tc in the form of ammoniumpertechnetate in 0.1M ammonium hydroxide solution was obtained fromAmershal International.

(i) A solution of ^(99m) Tc-pertechnetate (50 MCi˜18 GBq for animalstudies) were taken to dryness using a rotary evaporator. Sodium azide(˜20 mg) was added to the dry residue, followed by 10 ml of concentratedhydrochloric acid (sg 1.18). The solution was refluxed for 5 minutes tocomplete the reduction and destroy excess azide before being taken todryness in the rotary evaporator. 1 ml of ligand solution (PIPIDA 20mg/ml, pH 7, all others 5 mg/ml, pH 7) was added followed by 2 mlsaline. If necessary, the pH was adjusted to 6-7 by the additionof 0.1Msodium hydroxide. After filtration through a 0.22μ membrane filter, theradiopharmaceutical was ready for use.

(ii) An alternative labelling procedure is to perform the ligandreplacement in a non-aqueous solvent, such as in acetonitrile or ethanolsolution. By way of example, 10 ml of acetonitrile is added to the dryresidue after the azide reduction followed by 100 μl of the ligandsolution. After heating on a water bath for 10 minutes, the acetonitrileis removed in a rotary evaporator and the dried extract dissolved in 1ml of the ligand solution and 2 ml saline as before.

B. Animal Distribution Studies

0.05-0.1 ml of the preparation (1-2 mCi) was injected into the tail veinof Swiss mice (20-30 g) and the activity injected measured in anionization chamber. After injection, mice were allowed free access tofood and water. At each time interval studied, three mice weresacrificed by cervical dislocation and dissected. Organs were weighedand their activities measured in the ionisation chamber. The originalinjected activity was corrected for the activity found in the tail.Blood activity was calculated on the assumption that the overall bloodvolume represents 7% of total body weight.

C. Measurement of ^(99m) Tc Labelling

2 μL aliquots of each preparation were chromatrographed on Whatman No. 1paper in three solvent systems: saline, 70% methanol and methyl ethylketone. After development all papers were dried and scanned in aradiochromatogram scanner (Packard Model 7220/21). Where appropriate,peaks were cut from the strips and counted for ^(99m) Tc activity in agamma counter. All preparations contained less than 5% freepertechnetate.

D. UV Spectral Studies

Preparations were made using 300 μg ⁹⁹ Tc added as carrier for UVspectral studies. UV spectra were recorded on a Beckman Acta CIIspectrophotometer.

Pertechnetate was reduced using the following reduction systems:

(i) concentrated hydrochloric acid,

(ii) concentrated hydrochloric acid/potassium iodide,

(iii) concentrated hydrochloric acid/sodium azide,

(iv) concentrated hydrochloric acid/stannous chloride,

(v) concentrated hydrochloric acid/hypophosphorous acid,

(vi) concentrated hydrochloric acid/hydroxylamine hydrochloride.

The spectra of all samples were measured in hydrochloric acid solutionbefore and after heating on a hot plate.

UV spectra of the technetium complexes studied were obtained aftertaking the above solutions to dryness in a rotary evaporator anddissolving the dry residue in 1 ml ligand solution and 2 ml water. ThepH of the solution was adjusted to 7 using either 0.1M or 1M sodiumhydroxide.

E. RESULTS

The concentration of Tc in the solution used for uv absorptionmeasurements was determined by beta counting. 10 mL scintillation fluid(Aquassure-NEN) were added to 0.1 mL aliquots of the solution forcounting in a liquid scintillation spectrometer. Counting efficiency wasdetermined by counting aliquots of a pertechnetate solution standardizedby uv spectrophotometry. Quenching of the solution was checked byexternal standardization. An internal standard was added when a quenchcorrection was necessary.

(i) UV Spectral Studies (a) Hydrochloric Acid Studies

Addition of potassium iodide, hydroxylamine hydrochloride, orhypophosphorous acid to pertechnetate in hydrochloric acid followed byheating all result in the formation of Tc(IV). This is shown in FIG. 1by the characteristic UV absorption spectrum of TcCl₆ ²⁻ which has anabsorption maximumat 340 nm and a minor peak at 305 nm. Pertechnetateallowed to stand in concentrated hydrochloric acid in the cold, resultsin the production of TcOCl₅ ²⁻ in which Tc is in the Tc(v) oxidationstate. The UV spectrum however indicates that the TcOCl₅ ²⁻ producedalso contains Tc(IV), the proportion of which is increased by heating.Heating of pertechnetate in the presence of azide results in theformation of TcNCl₄ ⁻ characterised by an absorption maximum at 395 nm(=500 m² mol⁻¹) and free of contaminating Tc(IV).

(b) MDP Complexes

The spectra of TcN-MDP and Tc(IV)-MDP are shown in FIG. 2. When MDP wasadded to TcNCl₄ ⁻ at pH 5.5, a pink colored complex (λ_(max) -515 nm)was produced. On heating the pink color disappeared during a yellowcomplex with an adsorption maximum at 335 nm. The spectrum of thiscomplex was identical to that obtained by adding MDP to TcNCl₄ ⁻ andadjusting the pH to 10 (λ_(max) =335 nm, ε=21 m² mol⁻¹).

(c) DTPA Complexes

When prepared at room temperature, TcN-DTPA showed an absorption maximumat 505 nm (ε=105 m² mol⁻¹) (FIG. 3). On heating the peak disappearedproducing a complex with no significant absorption in the range 300-800nm. Tc^(IV) -DTPA produced by adding DTPA to TcCl₆ ²⁻ showed nosignificant absorption maximum in the uv-vis region.

(d) Cysteine Complexes

The spectrum of TcN-CYS showed no significant absorption in theuv-visible region that could be attributed to the complex.

(ii) Stability of dried TcNCl₄ ⁻ Preparation

Samples of the ^(99m) TcNCl₄ ⁻ preparation were taken to dryness in arotary evaporator and stored for 24 hours (a) in air at roomtemperature, (b) at 80° C., and (c) at room temperature and 100%humidity. They were then used to prepare ^(99m) TcN-DTPA by addition ofligand as before. No significant difference in chromatographic behaviouras measured by high performance liquid chromatography (HPLC) wasobserved in any sample indicating that the dry ^(99m) TcNCl₄ ⁻ reagentwas stable for up to 24 hours.

(iii) Animal Distribution Studies

(a) Results of biodistribution studies of ^(99m) TcN-MDP, ^(99m)TcN-DTPA and ^(99m) TcN-CYS are given in Tables 1-3. ^(99m) TcN-MDP wascharacterized by high blood pool activity and showed significant boneuptake. ^(99m) TcN-DTPA showed similar renal excretion behaviour to^(99m) Tc-DTPA(Sn) and was probably excreted by a similar mechanism.Blood pool activity however was higher than that observed for ^(99m)Tc-DTPA(Sn). ^(99m) Tc-cysteine showed rapid clearance with high renallocalization. Urinary excretion rates for the three ^(99m) TcN-complexesare given in Table 4. Excretion rates of ^(99m) TcN-MDP and ^(99m)TcN-DTPA are significantly less than that of the respective ^(99m)Tc(Sn)-complexes.

(b) Results of biodistrubition studies of ^(99m) Tc-GLUC, ^(99m)TcN-HEDP, ^(99m) TcN-HIDA and ^(99m) TcN-PIDIDA ae given in Tables 5-9.All preparations showed high blood pool activity with the ^(99m)TcN-GLUC and ^(99m) TcN-HEDP preparations clearing slightly faster than^(99m) TcN-PIPIDA and ^(99m) TcN-HIDA. (FIG. 4). Overall, ^(99m)TcN-GLUC, ^(99m) TcN-HIDA and ^(99m) TcN-HEDP showed comparable patternsof biological behaviour. ^(99m) TcN-PIPIDA differed from the otheragents in that it gave significantly higher activity in the intestines.With all preparations the clearance into the intestines took placeessentially in the first 30 minutes. The higher clearance of ^(99m)TcN-PIPIDA could be due to ^(99m) TcN-PIPIDA undergoing a slower rate ofexchange with serum proteins than ^(99m) TcN-HIDA.

(c) Results of biodistribution studies of ^(99m) TcN-DMSA are given inTable 10.

(iv) Stability studies

Stability studies of the dry ⁹⁹ TcNCl₄ ⁻ preparations have indicatedthat the agent is sufficiently stable to be prepared in a centrallaboratory or manufacturing site and distributed to users for use in thepreparation of ^(99m) TcN-labelled radiopharmaceuticals. Preparation ofthe ^(99m) TcN-radiopharmaceuticals in most cases takes place by simplydissolving the dry residue in a solution of the chelate.

^(99m) TcNCl₄ ⁻ may also be used to label ligands that are insolubleinwater or which may be unstable in aqueous solutions. It is possible toextract the ^(99m) TcN-activity from the dry salt residue into organicsolvents such as acetonitrile. Labelling may be performed in the organicsolvent which may then be removed by evaporation. Labelling with ^(99m)TcNCl₄ ⁻ takes place via a substitution mechaism and is expected to beless susceptible to the hydrolytic type reactions which occur with manyother labelling procedures. In addition, the presence of the nitridogroup modifies the chemistry of the Tc atom in that reactions with"soft" ligands are more favoured than when other reducing agents areused.

EXAMPLE 2 A. Preparation of ^(99m) TcN-labelled MAb (i) Tumor Cell Lines

Two tumor cell lines were used: once, the E3 clonal variant of thethymoma ITT(1)75NS (Hogarth, et al, 1982), which was obtained by threesuccessive rounds of cytofluorographic sorting of IGG(1)75NS cellsstained with monoclonal Ly-2 antibodies and selected for the mostfluorescent 1% of cells. The murine cell line E(3) was maintained invitro in DME supplemented with 10% heat inactivated newborn calf serum(Flow Laboratories, Sydney, Australia), 2 mM glutamine (CommonwealthSerum Laboratories, CSL, Melbourne, Australia), 100 I.U./ml penicillin(CSL) and 100 mg/ml streptomycin (Glaxo, Melb., Aust.). E3 cells werewashed twice in DMI (without additives) and twice in DME containing 0.5%BSA and used in the in vitro binding assays. The E3 cell line wasmaintained in vivo by the passaging of cells from ascites fluid producedin (B6×BALB/c)F₁ mice or from solid tumors which grow after thesubcutaneous injection of 10⁶ or 10⁷ cells. The second cell line usedwas a human colonic carcinoma, COLO 205 (Semple et al, 1978). It wasmaintained in cuture with TPMI containing the same additives. AdherentCOLO 205 cells were harvested with 0.125% trypsin (CSL) washed with RPMIand injected subcutaneously into nude mice, where tumors appeared afterthe injection of 2×10⁶ -1×10⁷ cells.

(ii) Monoclonal Antibodies (MAb)

Two monoclonal antibodies were used: (i) anti-Ly-2.1 (IgG2a), anantibody raised against the murine alloantigen Ly-2.1 (Hogarth et al,1982) and (ii) 250-30.6 (IgG2b), an antibody to human colonic secretoryepithelium (Thompson, et al, 1983). The monoclonal antibodies wereisolated from ascitic fluid by precipitation with 40% saturated ammoniumsulphate, followed by dissolution in 0.01M Tris buffer pH 8.0 andextensive dialysis against the same buffer. The crude antibodypreparations were further purified by affinity chromatography usingprotein-A Sepharose (Pharmacia) and then purity determined by gelelectrophoresis and antibody activity assayed by a rosetting test(Parish, et al, 1978).

(iii) ^(99m) Tc Labelling of Antibodies

^(99m) TcNCl₄ ⁻ was prepared as described above. For labelling the MAbwas first reduced with dithiothreitol (DTT) by adding 20 μl of DTT inPBS (115 mg/ml) to 200 μg of MAb (1 mg/ml in PBS) and standing themixture at room temperature for 30 minutes after which the reduced MAbwas separated from DTT by gel chromatography using 0.1M sodium acetatepH 4.0 as eluant on an 8 cm×1 cm column of Biogel P-6 (BioradLaboratories, Richmond, USA). The fractions (1 ml) containing theprotein peak were added to the dried ^(99m) TcNCl₄ ⁻ salt residue andthe mixture brought to pH 3.0 with 0.2M hydrochloric acid. After 2minutes at room temperature, 2 drops of 0.1M sodium phosphate was addedand the pH adjusted to 7 by the careful addition of sodium hydroxide (1Mor 0.1M). Purification of the labelled MAb was then achieved by gelchromatography with a Sephadex G-25 disposable column (PD-10, Pharmacia)equilibrated in PBS, 500 μl aliquots were collected and theradiolabelled protein peak identified by gamma counting.

B. RESULTS (i) In vitro stability and specificity of ^(99m) TcN-MAb

Three different specificity assays were performed. The first comparedthe activity of ^(99m) Tc labelled by Ly-2.1 MAb to thymocytes from themouse strains RF/J (Ly-2.1 positive and C57BL/6 (Ly-2.1 negative). A tenfold difference was observed in the binding of the labelled MAb to thespecific cells (RF/J) when compared to the negative cell controls(C57BL/6). The procedure used to couple ^(99m) TcNCl₄ ⁻ to MAb produceda stable complex which was shown to be stable and bind specificallyantigen positive target cells ten times greater than antigen negativecontrol cells.

In the second specificity assay two different MAb, one directed againstcolonic secretory epithelium (250-30.6) and the second the anti-Ly-2.1MAb, were labelled with ^(99m) Tc under identical coupling conditionsand the two complexes were tested for their ability to bind to themurine T cell thymoma E3, which is positive for the Ly-2.1 antigen butdoes not react with the anti-colon antibody. The anti-Ly-2.1 MAb boundten times more efficiently than the ^(99m) TcN-anti-colon complex. Thestability of the ^(99m) TcN-MAb was demonstrated here, where only theantibody reactive complex (^(99m) TcN-anti-Ly-2.1) showed increasedbinding on E3 target cells. The non-reactive complex (^(99m)TcN-anti-colon) exhibited a significantly reduced uptake ofradioactivity on the same target cells.

The stability of the TcN-MAb complexes was examined in a third assay.Aliquots of labelled MAb were stored at 4° C. overnight and thestability of the label was determined by binding the stored material tothymocytes. The labelled MAb were shown to be stable by their retentionof binding ability. RF/J thymocytes (Ly-2.1⁺) bound better than 10 foldmore labelled Ly-2.1 MAb than C57BL/6 thymocytes (Ly-2.1⁻). In threedifferent specificity assays in the in vitro stability of the ^(99m)TcN-MAb complexes was established and were shown to be chemicallystable, even when allowed to stand at 4° C. overnight, binding only toantibody reactive target cells.

(ii) Localization

The in vivo localization and biodistribution of TcN-MAb complexes werepreformed in separate studies. In the first, the mice were dissectednormal organs, tumors and an aliquot of whole blood were counted in agamma counter. The solid tissues were then weighed and the results wereused to calculate the localization ratio derived as follows: tissue(cpm/g)/blood (cpm/g). Two groups of 16 (C57BL/6×BALB/c)F₁ mice bearingthe E3 tumor (0.23-1.11 g) were injected i.v. via the tail vein, withone of two MAb (anti-Ly-2.1 or anti-colon carcinoma) labelled with^(99m) TcNCl₄ ⁻ under identical conditions. (Each mouse received 115 μCiTc and 10 μg MAb). In the data in Table 11, 4 mice from each group weresacrificed at different time intervals after injection (20, 30.5, 35hrs) and the distribution of the specific MAb (anti-Ly-2.1) wascalculated for the individual tissues and compared to the observeddistribution of the non-reactive MAb (anti-colon). After 20 hrs thetumor localization was observed to be 3 times greater for the specificMAb than that observed for the non-specific MAb. This ratio increased toapproximately 3.8 at 30.5 hrs, and 7.3 at 35 hrs after injection. It isimportant to point out that the E3 tumor was observed to have thehighest localization ratio (i.e. tissue (cpm/g)/blood (cpm/g)) in thegroup injected with the specific MAb (anti-Ly-2.1) with the liver,spleen and kidney localization ratio being below or similar to the bloodratio. However, with the non-specific antibody (250-30.6) the liver,spleen and kidney were observed to be higher than the blood ratio, withthe liver biodistribution ratio being 5 times greater than the bloodafter 30.5 hrs.

In the second study, a specific MAb (anti-Ly-2.1) was compared in twodifferent tumors. Nude mice bearing colo 205 xenografts were used as thenon-reactive tumor and the E3 as the positive tumor. The data wasobtained 20 hrs after the injection of the anti-Ly-2.1 labelled MAb(Table 12). The E3 thymoma (Ly-2.1⁺) was observed to take up 3 timesmore radioactivity tha the colo 205 xenografts (Ly-2.1⁻ H). The twobiodistribution studies illustrate a significant increased incorporationof radioactivity in the MAb reactive tumor when compared to the levelsof radioactivity in the blood and that incorporated in the other normaltissues.

                  TABLE 1                                                         ______________________________________                                        Biological Distribution of .sup.99m TcN-MDP in                                mice*                                                                         Tine after                                                                             % Injected Dose/Organ                                                Injection                                                                              30 min       60 min     120 min                                      ______________________________________                                        Heart    0.6(0)       0.4(0)     0.3(1)                                       Lung     2.9(5)       3.0(14)    1.9(15)                                      Liver    10.4(4)      9.4(8)     6.2(13)                                      Spleen   0.4(1)       0.3(1)     0.4(1)                                       Stomach  1.1(1)       0.8(2)     1.2(11)                                      Kidneys  6.9(27)      3.3(5)     2.6(7)                                       Intestines                                                                             4.3(4)       4.1(6)     6.1(13)                                      Femurs   0.3(0)       0.3(0)     0.3(0)                                       Blood    26.5(46)     18.8(18)   13.5(40)                                            % Injected Dose/Gram Organ                                             Heart    4.2(2)       2.4(4)     2.0(4)                                       Liver    5.7(3)       4.7(5)     3.8(1)                                       Kidneys  13.0(54)+    5.6(11)    5.8(18)                                      Femurs   1.7(2)       1.4(2)     1.5(1)                                       Blood    13.0(22)     8.1(3)     6.7(19)+                                     ______________________________________                                         Standard deviation of last significant digit(s) in brackets.                  *n = 3 unless otherwise stated                                                + 2 mice                                                                 

                  TABLE 2                                                         ______________________________________                                        Biological Distribution of .sup.99m TcN-DTPA in                               mice*.                                                                        Time After                                                                             % Injected Dose/Organ                                                Injection                                                                              30 min       60 min     120 min                                      ______________________________________                                        Heart    0.1(0)       0.1(0)     0.1(0)                                       Lung     0.7(1)       0.7(2)     0.4(1)                                       Liver    1.7(2)       1.5(3)     1.1(2)                                       Spleen   0.1(0)       0.1(0)     0.1(0)                                       Stomach  1.1(2)       1.3(4)     0.9(0)                                       Kidneys  1.5(1)       1.3(2)     0.8(0)                                       Intestines                                                                             2.1(4)       2.0(3)     2.1(2)                                       Femurs   0.1(0)       0.1(0)     0.1(0)                                       Blood    6.2(8)       4.0(5)     2.4(1)                                              % Injected Dose/Gram Organ                                             Heart    0.6(0)       0.5(1)     0.4(1)                                       Liver    0.8(1)       0.7(1)     0.6(1)                                       Kidneys  2.5(1)       2.2(4)     1.5(2)                                       Femurs   0.5(1)       0.4(1)     0.4(1)                                       Blood    2.8(5)       1.8(2)     1.2(0)                                       ______________________________________                                         Standard deviation of last significant digit(s) in brackets.                  * n = 3                                                                  

                  TABLE 3                                                         ______________________________________                                        Biological Distribution of .sup.99m TcN-CYSTEINE                              in mice.*                                                                     Time After                                                                             % injected Dose/Organ                                                Injection                                                                              30 min       60 min     120 min                                      ______________________________________                                        Heart    0.1(0)       0.1(0)     0.1(0)                                       Lung     0.8(2)       0.5(1)     0.4(1)                                       Liver    2.7(5)       1.6(1)     1.3(1)                                       Spleen   0.1(0)       0.1(0)     0.1(0)                                       Stomach  0.4(1)       0.3(3)     0.3(1)                                       Kidneys  4.4(5)       3.4(5)     2.8(2)                                       Intestines                                                                             5.3(8)       3.4(1)     4.0(3)                                       Femurs   0.2(0)       0.1(1)     0.1(0)                                       Blood    4.9(7)       2.8(4)     1.8(1)                                              % Injected Dose/Gram Organ                                             Heart    0.7(1)       0.5(2)     0.4(2)                                       Liver    1.3(2)       0.8(2)     0.6(1)                                       Kidneys  7.4(11)      6.2(9)     4.8(5)                                       Femurs   0.8(1)       0.6(3)     0.2(1)                                       Blood    2.1(2)       1.3(1)     0.8(1)                                       ______________________________________                                         Standard deviation of last significant digit(s) in brackets.                  * n  3                                                                   

                  TABLE 4                                                         ______________________________________                                        Urinary Clearance of .sup.99m TcN-MDP, .sup.99m TcN-DTPA                      and .sup.99m TcN-CYS*                                                                       % Retained Activity                                             .sup.99m TcN-MDP                                                                              .sup.99m TcN-DTPA                                                                          .sup.99m TcN-CYS                                 ______________________________________                                        30 min 71.9(43)     24.8(42)     35.8(43)                                     60 min 51.4(14)     21.4(31)     18.7(28)                                     120 min                                                                              44.7(57)     14.6(6)      15.7(15)                                     ______________________________________                                         Standard deviation of last significant digit(s) in brackets.                  * n = 3                                                                  

                  TABLE 5                                                         ______________________________________                                        Biological Distribution of .sup.99m TcN-GLUC in mice.                         % Injected Dose/Organ                                                         Time After                                                                    Injection 30 min       60 min   120 min                                       ______________________________________                                        Heart     0.7(0.1)     0.7(0.1) 0.4(0.1)                                      Lung      2.2(0.6)     3.0(0.5) 1.4(0.2)                                      Liver     6.4(1.6)     6.2(1.7) 7.1(2.1)                                      Spleen    0.3(0.1)     0.3(0.1) 0.2(0.1)                                      Stomach   1.4(0.4)     1.5(0.4) 1.6(0.7)                                      Kidneys   4.7(0.6)     3.9(0.3) 3.4(0.1)                                      Intestines                                                                              5.4(0.7)     4.1(0.8) 7.0(1.4)                                      Femurs    0.3(0.0)     0.3(0.0) 0.2(0.1)                                      Blood     25.7(2.3)    21.7(3.6)                                                                              12.7(1.9)                                     % Injected Dose/Gram Organ                                                    Heart     4.5(0.7)     4.6(1.0) 2.6(0.6)                                      Liver     3.3(0.8)     3.1(0.9) 3.3(0.8)                                      Kidneys   8.4(1.0)     6.6(0.7) 5.6(0.6)                                      Femurs    1.7(0.1)     1.3(0.1) 0.8(0.2)                                      Blood     12.0(1.0)    9.5(1.8) 5.2(0.3)                                      ______________________________________                                         Standard deviation in brackets.                                               n = 3.                                                                   

                  TABLE 6                                                         ______________________________________                                        Biological Distribution of .sup.99m TcN-HEDP in mice.                         % Injected Dose/Organ                                                         Time After                                                                    Injection 30 min       60 min   120 min                                       ______________________________________                                        Heart     0.5(0.0)     0.3(0.0) 0.3(0.0)                                      Lung      2.5(0.6)     1.8(0.3) 1.5(0.4)                                      Liver     12.1(0.3)    9.0(1.0) 7.7(0.5)                                      Spleen    0.6(0.1)     0.3(0.0) 0.3(0.1)                                      Stomach   0.6(0.1)     0.7(0.1) 0.6(0.1)                                      Kidneys   4.1(0.2)     3.2(0.2) 3.0(0.2)                                      Intestines                                                                              6.9(0.5)     7.0(0.2) 6.8(0.6)                                      Femurs    0.5(0.0)     0.5(0.1) 0.4(0.1)                                      Blood     28.0(5.0)≠                                                                           16.2(2.0)                                                                              13.2(1.0)≠                              Heart     4.3(0.5)     2.4(0.3) 2.3(0.2)                                      Liver     8.1(0.8)     5.5(0.7) 4.8(0.4)                                      Kidneys   9.1(0.4)     6.5(0.4) 6.1(0.3)                                      Femurs    3.4(0.5)     3.1(0.8) 2.7(0.6)                                      Blood     16.2(1.0)≠                                                                           7.3(1.1) 7.7(0.5)≠                               ______________________________________                                         Standard deviation in brackets.                                               n = 3 unless otherwise stated.                                           

                  TABLE 7                                                         ______________________________________                                        Biological Distribution of .sup.99m TcN-HIDA in mice.                         % Injected Dose/Organ                                                         Time After                                                                    Injection 30 min       60 min   120 min                                       ______________________________________                                        Heart     0.4(0.1)     0.4(0.0) 0.2(0.0)                                      Lung      2.3(0.3)     2.0(0.3) 1.2(0.3)                                      Liver     7.2(1.4)     6.7(1.2) 3.6(0.2)                                      Spleen    0.4(0.1)     0.3(0.0) 0.1(0.0)                                      Stomach   1.1(0.2)     2.0(0.4) 1.0(0.0)                                      Kidneys   3.5(0.2)     3.4(0.2) 2.1(0.0)                                      Intestines                                                                              6.8(1.0)     9.2(0.4) 6.9(0.2)                                      Femurs    0.3(0.1)     0.4(0.0) 0.2(0.0)                                      Blood     25.8(1.6)    20.6(1.3)                                                                              16.3(2.2)                                     % Injected Dose/Gram Organ                                                    Heart     2.9(0.5)     2.7(0.4) 1.6(0.1)                                      Liver     4.2(0.8)     3.9(0.6) 2.1(0.1)                                      Kidneys   6.9(0.5)     6.7(0.4) 4.2(0.1)                                      Femurs    2.0(0.4)     2.3(0.1) 1 0(0.1)                                      Blood     13.6(0.6)    10.8(0.6)                                                                              8.7(1.1)                                      ______________________________________                                         Standard deviation in brackets.                                               n = 3.                                                                   

                  TABLE 8                                                         ______________________________________                                        Biological Distribution of .sup.99m TcN-PIPIDA in mice. -% Injected           Dose/Organ                                                                    Time After                                                                    Injection 30 min       60 min≠                                                                          120 min                                       ______________________________________                                        Heart     0.4(0.0)     0.3(0.1) 0.4(0.1)                                      Lung      2.3(0.3)     1.8(0.6) 1.8(0.2)                                      Liver     14.6(1.0)    16.2(6.1)                                                                              10.1(1.0)                                     Spleen    0.2(0.1)     0.3(0.0) 0.2(0.0)                                      Stomach   0.7(0.1)     1.0(0.0) 0.8(0.1)                                      Kidneys   4.6(0.5)     7.6(5.6) 3.3(0.1)                                      Intestines                                                                              12.8(1.5)    12.0(0.8)                                                                              14.9(2.1)                                     Femurs    0.3(0.0)     0.2(0.0) 0.2(0.0)                                      Blood     26.8(3.0)    23.2(0.4)                                                                              20.0(3.7)                                     % Injected Dose/Gram Organ                                                    Heart     3.4(0.2)     2.1(0.8) 2.8(0.5)                                      Liver     8.8(0.6)     8.9(3.1) 5.8(0.8)                                      Kidneys   9.4(0.6)     14.1(7.2)                                                                              6.3(0.2)                                      Femurs    2.0(0.3)     1.1(0.1) 1.0(0.2)                                      Blood     14.8(1.9)    11.5(0.7)                                                                              10.2(1.5)                                     ______________________________________                                         Standard deviation in brackets.                                               ≠n = 3.                                                            

                                      TABLE 9                                     __________________________________________________________________________    Urinary Clearance of .sup.99m TcN-GLUC, .sup.99m TcN-HEDP,                    .sup.99m TcN-HIDA and .sup.99m TcN-PIPIDA                                     % Retained Activity                                                           .sup.99m TcN-GLUC                                                                           .sup.99m TcN-HEDP                                                                     .sup.99m TcN-HIDA                                                                     .sup.99m TcN-PIPIDA                             __________________________________________________________________________    30 min                                                                              72.6(4.2)                                                                             68.8(9.5)                                                                             74.9(5.8)                                                                             79.5(1.2)                                       60 min                                                                              59.3(6.4)                                                                             52.2(1.2)                                                                             66.3(0.9)                                                                             73.2(2.7)                                       120                                                                              min                                                                              45.6(5.6)                                                                             47.2(4.5)                                                                             54.7(1.1)                                                                             62.0(2.5)                                       __________________________________________________________________________     Standard deviation in brackets.                                               n = 3.                                                                   

                  TABLE 10                                                        ______________________________________                                        Biological distribution of .sup.99m TcN-DMSA                                  % Injected Dose/Organ                                                         Time after                                                                    Injection 30 min       60 min   120 min                                       ______________________________________                                        Heart     0.3(0.0)     0.3(0.0) 0.2(0.0)                                      Lung      1.6(0.1)     1.1(0.2) 1.1(0.2)                                      Liver     4.3(0.1)     3.9(0.3) 3.4(0.2)                                      Spleen    0.4(0.1)     0.4(0.1) 0.3(0.0)                                      Stomach   0.6(0.1)     0.7(0.2) 0.5(0.1)                                      Kidney    10.3(0.5)    12.3(0.8)                                                                              17.1(0.7)                                     Intestines                                                                              5.3(0.7)     5.1(0.4) 4.8(0.5)                                      Femurs    0.5(0.1)     0.5(0.0) 0.4(0.1)                                      Blood     18.0(1.7)    13.5(0.9)                                                                              9.5(0.5)                                      Urine     39.9(11.4)   57.8(4.4)                                                                              65.3(4.3)                                     % Injected Dose/Gram Organ                                                    Heart     1.8(0.1)     1.8(0.2) 1.2(0.0)                                      Liver     2.0(0.2)     2.1(0.3) 1.7(0.2)                                      Kidney    16.4(0.7)    22.2(1.4)                                                                              29.0(2.9)                                     Femurs    2.0(0.5)     2.4(0.2) 1.9(0.4)                                      Blood     7.1(0.8)     6.4(0.3) 4.1(0.2)                                      ______________________________________                                         Standard Deviation in brackets                                                n = 3.                                                                   

                  TABLE 11                                                        ______________________________________                                        Biodistribution Ratio Tissue (cpm/g): Blood (cpm/g)                           Specific MAb (anti-Ly-2.1)/Non-specific Mab (anti-colon)                             Time after Injection                                                   Organ     20 hrs      30.5 hrs    35 hrs                                      ______________________________________                                                 α2.1                                                                            30.6    α2.1                                                                          30.6  α2.1                                                                          30.6                               Blood    1.00    1.00    1.00  1.00  1.00  1.00                               Tumor    1.23    0.40    2.60  0.68  3.48  0.48                               (0.23-1.11 g)                                                                 Stomach  0.08    0.12    0.12  0.17  0.07  0.12                               Spleen   0.59    1.12    0.56  1.83  0.71  2.09                               Kidney   0.77    1.48    0.94  2.14  0.92  2.50                               Heart    0.33    0.39    0.19  0.17  0.33  0.19                               Liver    0.84    0.27    1.02  3.26  1.20  5.19                               Lung     0.38    0.22    0.86  0.66  0.36  0.60                               Intestine                                                                              0.09    0.16    0.15  0.20  0.09  0.19                               Tail     1.45    0.90    0.78  1.92  1.14  1.66                               ______________________________________                                    

                  TABLE 12                                                        ______________________________________                                        Biodistribution Ratio Tissue (cpm/g): Blood (cpm/g)                                  ITT(1)E3  COLO 205  E3/COLO 205                                        ______________________________________                                        Blood    1.0         1.0       1.0                                            Tumor    1.23        0.30      4.10                                                    (.39-1.11 g)                                                                              (0.5-1.5 g)                                              Stomach  0.08        0.2       0.04                                           Spleen   0.59        0.60      0.98                                           Kidney   0.77        0.76      1.01                                           Heart    0.33        0.39      0.85                                           Liver    0.84        0.56      1.50                                           Lung     0.38        0.42      0.90                                           Intestine                                                                              0.09        0.15      0.60                                           ______________________________________                                    

REFERENCES

1. Castronovo, F. P., J. Nucl. Med. 15, 127 (1974).

2. Callery, P. S., Faith, W. C., Loberg, M. D., Fields, A. T., Harvey,E. B. and Cooper, M. D. J. Med. Chem, 19, 962 (1976).

3. Deutsch, E., Libson, K., Jurisson, S. et al: Technetium chemistry andtechnetium radiopharmaceuticals. Prog. Inorg. Chem 30: 75-139, 183.

4. Deutsch, E., Barnett, B. L.: Synthetic and structural aspects oftechnetium chemistry as related to nuclear medicine, in: InorganicChemistry in Biology and Medicine (ed Martel A. E.), ACS Symp. SeriesNo. 150, Washington, Amer. Chem. Soc. 1980, pp. 103-119.

5. Hogarth, P. M., Henning, M. M. and McKenzie, I. F. C. Thealloantigenic phenotype of radiation induced thymomas in the mouse. J.N. C. I. 1982; 69: 619-626.

6. Hogarth, P. M., Edwards, J., McKenzie, I. F. C., Goding, J. W. andLiew, F. Y. Monoclonal antibodies to murine Ly-2.1 surface antigen,Immunology 1982; 46: 135-144.

7. Parish, C. R. and McKenzie, I. F. C. A sensitive rosetting method fordetecting subpopulations of Lymphocytes which react with alloantisera.J. Immunol. Methods 1978; 20: 173-183.

8. Rhodes, B. A. and Burchiel, S. W. Radiolabelling of Antibodies withTechnetium 099m. Radioimmunoimaging and Radioimmunotherapy. EditorsBurchiel, S. W. and Rhodes, B. A. Elsevier publishing Co. 1983, p. 207.

9. Semple, T. U., Quinn, L. A., Woods, L. F. and Moore, G. E. Tumor andLymphoid cell lines from a patient with carcinoma of the colon for acytoxicity model. Cancer Res. 1978; 38: 1345-1355.

10. Thompson, C. H., Jones, S. L., Pihl, E. and McKenzie, I. F. C.Monoclonal antibodies to human colon and colorectal carcinoma. Br. J.Cancer 1983; 47: 595-605.

11. Tubis, M., Krishnamurthy, G. T., Endow, J. S., Blahd, W. H.(1975)-^(99m) Tc-Penicillamine complexes:In:Subramanian G., Rhodes, B.A., Cooper, J. F., Sodd, V. J. (eds) "Radiopharmaceuticals". The Societyof Nuclear Medicine Inc., New York, pp. 55-62.

We claim:
 1. A compound of the formula I:

    R.sup.+ [.sup.99m TcNX.sub.4 ].sup.- I

wherein R⁺ represents a catio and X represents a halogen group.
 2. Acompound according to claim 1, wherein R⁺ represents a sodium or anotheralkali metal or ammonium cation, and X represents a chlorine or bromineatom.
 3. A process for preparing a compound of the formula I defined inclaim 1, which comprises reaction of a compound of the formula II:

    R.sup.+ [.sup.99m TcO.sub.4 ].sup.- II

wherein R⁺ is as defined in claim 1, with an azide compound in thepresence of a hydrohalic acid.
 4. A process according to claim 3,wherein R⁺ represents a sodium or another alkali metal or ammoniumcation, the azide compound is sodium azide, and the hydrohalic acid ishydrochloric or hydrobromic acid.